Length-dependent recoil separation of long molecules

ABSTRACT

Separation of long molecules by length is obtained by forcing such molecules to traverse a boundary between a low free-energy region and a high free-energy region. In one embodiment, the high free-energy region is a dense pillar region or other structure formed on a semiconductor substrate. One or more membranes are used in further embodiments. The low free-energy region is a larger chamber formed adjacent the high free-energy region. A recoil phase allows longer molecules not fully driven into the high free-energy region to recoil into the low free-energy region. In a further variation, the high free-energy region is a membrane having nanoscale holes.

This application claims the benefit under 35 U.S.C. § 119(e) of priorityto U.S. Provisional Patent Application Ser. No. 60/277,136, filed Mar.19, 2001, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to separation of long molecules, and inparticular to Length-Dependent recoil separation of long molecules.

BACKGROUND OF THE INVENTION

In its native environment, the structure of DNA is strongly influencedby the presence of cellular components such as histones, which bind theDNA helping to stabilize it and assist with its organization during celldivision. The familiar shape of chromosomes is due primarily to acomplex condensation process caused by interactions between DNA andthese nuclear proteins which bind to it.

Stripped of all of these other materials, DNA behaves quite differently.The conformation and motion of DNA by itself in solution are dominatedby constant thermal bombardment and statistical effects. On thenanometer scale, DNA is a stiff molecule. The stiffness of the moleculeis described by a parameter called the persistence length. Despite therelative stiffness of DNA, for sufficiently long molecules it tends toform a spherical “blob”, which is the technical term used by the polymerdynamics community to refer to the conformation of a polymer in freesolution. The size of this blob depends on the length of the DNAmolecule and the persistence length. DNA is a highly charged molecule,and because the normal aqueous buffer solutions used with DNA have highdensities of free ions, the electrostatic forces on DNA molecules arestrongly influenced by the screening effects of charges calledcounterions which are attracted to the DNA molecule. Because the DNAmolecules are charged, they move under the influence of an electricfield, but the details of this motion are complicated by hydrodynamicinteractions between molecules and their counterions. It turns out thatthe mobility (the ratio of the drift velocity to the applied field) doesnot depend on the size of the molecule. This is the reason gels are usedfor separating DNA—the interactions between the DNA and the polymerchains in the gel are responsible for the mobility differences betweendifferent sized molecules. Gels fail, however, when the DNA chains getvery long. The field-induced elongation of the molecule in the axialdirection spoils the length dependence of the mobility and for longchains there is no separation.

It is possible to extend the operational range of gel electrophoresis byreducing the field, but the time required for a separation quickly growsto unreasonable durations. A host of strategies for avoiding this limithave been developed, but still the separation of long DNA strands by gelelectrophoresis is a time-consuming process.

There is a need for a separation technique that does not exhibit adverselength dependence. There is a further need for a long moleculeseparation technique that can be done in a short time.

SUMMARY OF THE INVENTION

Separation of long molecules by length is obtained by forcing suchmolecules to traverse a boundary between a low free-energy region and ahigh free-energy region. The boundary between these two regions createsa “free-enegry barrier” which impedes the passage of molecules in thedirection from low free-energy to high free-energy. In one embodiment,the high free-energy region is a dense pillar region which induces areduction in entropy of the molecules. The structure is formed on asemiconductor substrate. The low free-energy region is a larger chamberor structure formed adjacent the high free-energy region.

In one embodiment, the low free-energy region comprises a chamber, andthe high free-energy region comprises a region of dense pillars. The lowfree-energy region corresponds to a high entropic region, and the highfree-energy region corresponds to a low entropic region, because of therelationship between entropy and free energy F=U−TS. The low and highfree-energy regions are created by opposing electric fields with adiscrete boundary in a further embodiment. Other types of structuresalso provide a sufficient entropic barrier, such as fritglass pate andmesopourous materials. The barrier is two-dimensional for applicationssuch as a semiconductor chip products and three dimensional for furtherapplications.

In one embodiment the high free-energy region comprises nanoscale holesin a membrane. Such membranes are commercially available and aremanufactured by techniques such as the track-etch technique known in theart of membrane filter manufacture. In this technique, a polycarbonatemembrane is subjected to bombardment by high-energy ionic nuclearfragments. These fragments cause chemical changes to the polymer matrixin the vicinity of the path along which the ion travels. These chemicalchanges change the solubility of the matrix in some etchants, such aspotassium hydroxide, allowing the tracks to be developed into smallholes. These holes can be made very regular in size and shape, and canrange in size from 10 nm in diameter or smaller up to several microns.In the present embodiment, the membrane is contacted with the medium inwhich the molecules are suspended and the force on the molecules isdirected perpendicular to the membrane. The free-energy barrier isformed by the lower entropy of molecules situated inside the pores inthe membrane. A further embodiment is several of these membranes stackedtogether to form an array of juxtaposed barriers in which separationoccurs. An open fabric of fine threads or a surface texture modificationof the membrane itself restricts contact between adjacent membranes tothose areas elevated by the texturing.

If one applies a force directed to push a molecule towards a region ofhigh free-energy, there will exist a critical force below which therewill be insufficient impetus to drive molecules into this highfree-energy region. Above the critical force the molecules will bereadily driven into the high free-energy region. It should be noted thatthe transition is not abrupt, as thermal fluctuations can occasionallycause molecules to make the transition even when the force issubcritical.

In an embodiment of the invention, a subcritical force is applied topush molecules upstream from the high entropic region to a boundarybetween the high and low entropic regions, where they will stop for lackof sufficient force. A pulse of supercritical force is applied for aduration. During this pulse, molecules undergo a period of transitionfrom the low free-energy region into the high free-energy region. For aparticular duration, some molecules will be short enough to be promotedentirely into the high free-energy region, while some molecules will besufficiently long that they will fail to be completely promoted into thehigh free-energy region, and some portion of these longer molecule willremain in the low free-energy region at the end of the pulse. Theduration and voltage level of the pulse can be selected dependent on thelength of molecule desired to be separated. In one embodiment of theinvention, the motive force is an electric field. Other forces may alsobe used such as pressure, gravity and centrifugal forces.

When the pulse is complete, most molecules that are not completelywithin the high free-energy region extract themselves back into the lowfree-energy in a recoil phase, while most molecules with no portion leftin the low free-energy region remain in the high free-energy region.When the recoil phase is complete, the molecules remaining in the highfree-energy region are generally of shorter length. Thus molecularseparation is effected. In a further embodiment, a retrograde pulse isprovided to enhance extraction.

The process is repeated in one embodiment with progressively longer andlonger pulses to observe and separate a broad range of lengths. In afurther embodiment, repetitive pulses of the same length are seriallystaged to amplify selectivity in a narrower range of lengths.

In further embodiments, a lateral or longitudinal AC voltage is appliedduring the recoil phase to speed the extraction of longer molecules.Further reducing the entropy of the low entropy region is done tofurther speed the extraction or recoil of longer molecules. Thetemperature of the system may also be increased with a similar effect ofincreasing the energy difference between the two regions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a timeline graphic representation of a molecule separationprocess utilizing a force barrier.

FIG. 2 is a block diagram representation of obstacles used to create theforce barrier of FIG. 1.

FIG. 3 is a graphic representation of entropic recoil over time.

FIG. 4 is a block representation of an apparatus for performing theprocess of FIG. 1.

FIG. 5 is a perspective view of a porous membrane used to create a forcebarrier.

FIG. 6 is a perspective view of a porous membrane having additionalsurface features.

FIG. 7 is a perspective view of a plurality of juxtaposed porousmembranes having addition surface features.

FIG. 8 is a time sequence illustrating molecule movement throughdifferent phases of application of force.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, reference is made to the accompanyingdrawings that form a part hereof, and in which is shown by way ofillustration specific embodiments in which the invention may bepracticed. These embodiments are described in sufficient detail toenable those skilled in the art to practice the invention, and it is tobe understood that other embodiments may be utilized and thatstructural, logical and electrical changes may be made without departingfrom the scope of the present invention. Values for parameters describedmay be further modified, but may not function as well as the valuesprovided in the description. The following description is, therefore,not to be taken in a limited sense, and the scope of the presentinvention is defined by the appended claims.

A separation cycle using a free-energy barrier, and pulses of force isrepresented in FIG. 1. In this embodiment, the energy barrier comprisesa region of dense pillars or a porous membrane 110 that provides an areaof high free-energy. Two molecules, a long molecule 112 and a shortermolecule 114 are shown on a low free-energy side of the free-energybarrier. The pillar region 110 is shown at a number of different timescorresponding to different phases in a program of various applied forcelevels. At a first time, 116, minimal if any force is applied across thepillar region. The molecules 112 and 114 are located on the lowfree-energy side of the force barrier. At a second time, 122, themolecules are represented in the same portions of the pillar region 110,but a gathering force 120 is provided across the pillar region causingthe molecules to gather at the force barrier. A higher force pulse 125is applied at 128, causing the long molecule to become partially driveninto the dense pillar region. A portion of the long molecule 112 stillremains outside the pillar region at the conclusion of the pulse 125.The shorter molecule 114 has been driven fully into the dense pillarregion.

At the conclusion of the higher force pulse 125, the force drops backdown to its initial level identified at 130 at time 134. During thislower force level, the longer molecule recoils back to the area lowerfree-energy, completing its recoil at time 136. The process may berepeated one or more times as indicated at 140.

The gather stage at 122 utilizes a subcritical force to push themolecules, such as DNA molecules upstream from the dense pillar regionagainst the boundary, where they are stopped because the field isinsufficient to overcome the free-energy barrier.

The drive pulse 125 is a short pulse of high force strength(supercritical force), well over the critical value to start driving themolecules into the dense pillar region. In this embodiment the obstaclescause the molecules reptate into the array of dense pillars. After atime, some of the molecules 114 have completely entered the dense pillarregion, while others 112 have some portion still in the low free-energyregion.

The recoil time is a time when the molecules are actually separatedaccording to size. If a molecule is not completely in the dense pillarregion, it extracts itself due to the forces induced by the differencein free-energy between the two regions. Molecules which have no partleft in the low free-energy region remain stationary following the shorthigh field pulse. In one embodiment, the process is repeated iterativelywith progressively longer and longer pulse durations suitable for abroad range of molecule lengths. It can also be repeated with the sameconditions to maximize the sensitivity in a smaller range of moleculelength. The barriers can be serially staged to amplify the selectivity.

Forces are felt by a molecule whenever it traverses the boundary betweenregions of high free-energy and low free-energy. It causes the moleculeto recoil from the region of high free-energy to the region of lowfree-energy. During the recoil phase of the motion, there are no appliedfields in an embodiment of the invention. Thus, the only force acting toimpede the recoil motion is the viscous drag between the molecule andthe walls of the device. Longer molecules require more time to recoilfrom the dense pillar region. The recoil time increases like the squareof the length because longer molecules have greater friction and theyhave farther to go. In one embodiment, pore size (the distance betweenpillars or the size of pores in a membrane) is decreased to furtherreduce the entropy in the confined region and increase the rate ofrecoil. In another embodiment the distance between the floor and ceilingof the chamber is made smaller in the high-free energy region to furtherreduce the entropy of molecules in the confined region and increase therate of recoil.

When a DNA molecule is trapped at an abrupt entropic barrier, the radiusof gyration is much smaller than the length of the onset of the barrier,so it is not appropriate to treat the molecule as a point particle, witha potential modified by the entropy. Where the onset of the barrier isabrupt, it is necessary to consider the details of the conformation of amolecule as it traverses the boundary.

Consider the case of a DNA strand in a tube with an abrupt constriction.Suppose the molecule is being driven by an electric field. When themolecule reaches the constriction, it must surmount the free energeticbarrier presented by the change in entropy between the wide region andthe thin region.

For sufficiently long molecules the entropy is approximately anextensive quantity. That is to say, if a molecule of length, 1, in aparticular region has an entropy S, then a molecule of length 2*1 willhave an entropy of approximately 2*S. Moreover, for a molecule partoccupying the thick region and part occupying the thin region, theentropy will be approximately a weighted sum of the characteristicentropies of each region.

The most expedient method for generating a value for the entropic forceis a Monte Carlo simulation. It turns out that the values for theabsolute entropy for a molecule in either region are not required, justthe difference in entropy between the two.

A two dimensional planar array of obstacles/pillars is shown in FIG. 2generally at 210. In one embodiment, the persistence length of thepolymer is about 65 μnm. The distance between a floor 220 and a ceiling230 of the dense pillar region is about 80 nm. In this case, it isexpedient to treat the contour of the molecule as two-dimensional. Thisis done because the floor-to-ceiling height is on the same order as thepersistence length. For stiff polymers like DNA, self-avoidance can beignored because self-interactions are rare. For DNA this is anacceptable approximation, because the persistence length is more than 30times larger than the diameter of the molecule. In one embodiment thehigh free-energy region in FIG. 2 comprises nanoscale holes in amembrane as described further below.

A dense pillar region is shown at 310 in FIG. 3. A molecule is shownpartially driven into the dense pillar region at 320 followingapplication of a pulse of force. At 330, the molecule is shown partiallyrecoiled from the dense pillar region, and at 340, the molecule is shownjust about fully recoiled after a lapse of time. The time it takes torecoil is dependent on the length of the molecule. Reducing the recoiltime can be done by applying a lateral alternating current fieldcalculated to make the molecule move sideways with respect to thepillars a distance approximately equal to the pillar separation.However, the AC field may be applied to cause the molecule to morefurther or shorter distances. In one embodiment, the AC field isapproximately 100 hertz, designed to make the molecule move at a speedof approximately 10 micrometers per second. A longitudinal AC field mayalso be applied in a similar manner.

In one embodiment, the gathering field is ¼ to ½ volts per centimeter todrive the molecules to the boundary. The drive pulse is approximatelyfive volts per centimeter in one embodiment for DNA molecules, but ishighly dependent on the pillar spacing. The pillar spacing for DNAmolecules was 40 to 70 nanometers in one embodiment, but also may bemuch larger with smaller effect. A pillar spacing of ½ micron or morealso provides some effect for DNA molecules. If the drive pulse is toolow, the minimum force to cross the barrier will not be obtained, andthere will be no separation of the molecules. If the drive pulse is toohigh, it may cause initiation of hernias along the molecule, causing thelonger molecules to also be completely driven across the barrier.

In further embodiments, the pulse of applied force is provided bypressure, gravity, centrifugal force, or some other force that causesmolecules to move. The force barrier in further embodiments comprisesfritglass plate or mesopourous materials. A fritglass plate comprisesnanoparticles fused together to form a glassy sandstone type material. Amesopourous material comprises naturally occurring zeolytes and manmadenanostructures having random nanostructure pores. In yet a furtherembodiment, an electrical energy barrier is used. The electrical enegryis uniform on both sides to form a tightly defined boundary.

The use of a force barrier in combination with pulses of force followedby a recoil phase may also be used on other biologicals, includingproteins. In the case of proteins, the pillar spacing and drive pulseare modified consistent with the persistent length of the desiredmolecule. It is also used for washing and separation and rinsing infurther embodiments without allowing the desired molecule to escape. Itshould be noted that these are just alternative forms of separatingmolecules and portions of molecules.

In a further embodiment, multiple spaced free-energy barriers areformed. When cycled with alternating drive pulses and recoil phases, amolecule length resolution is obtained that resembles pulse field gelelectrophoresis, but performs better. In one embodiment, the number ofbarriers is approximately 100.

FIG. 4 is a block representation of an apparatus 410 for separatingmolecules by use of a free-energy barrier. The free-energy barrier isrepresented at 415 as a dense pillar region supported on a structure417, which is glass in one embodiment or other supporting material thatis electrically isolated from the force barrier 415. A pair ofreservoirs for molecules in a buffer solution are represented at 420 and425, with corresponding covers 430, 435. The covers are made of glass inone embodiment to prevent evaporation. Each reservoir contains anelectrode 440 and 445 that is in contact with the buffer solution toprovide an electric field between the two reservoirs 420 and 425. Thebuffer solution is an electrolytic solution in one embodiment, andextends throughout the pillars of the force barrier 415. A device forviewing molecules from a distance is indicated at 450. Device 450comprises a fluorescence microscope in one embodiment, which allows thedirect visualization of individual DNA molecules as they interact withthe boundary between the dense pillar region and the free region.

The DNA samples for one embodiment are extracted from phages, primarilyT2 and T7 phage DNA. The T2 DNA is more than four times as long as theT7 with lengths of 164 and 37.9 kbp, respectively. This translates to afully stretched length of 50.8 um and 11.7 um, respectively. One of thestandard buffer solutions for working with DNA is a mixture of 89 mMTris-borate and 2 mM EDTA which is abbreviated TBE. The solution isusually shipped ten times concentrated, a form frequently called“10×TBE”. In one embodiment of the invention, a solution at 5 timeshigher concentration than usual, referred to as 5×TBE. The higher saltconcentration helps suppress electro-endoosmosis which is sometimescalled electro-osmosis or electrokinetic flow, in which an electricfield applied across a fluid-filled capillary causes flow in thecapillary. It is caused by donation of charges from the capillary wallto the fluid, which is then no longer net-electrically neutral, andresponds to the field by moving.

A fluorescent dye YOYO-1 is used in the solution. It is so named for itsconformation, which is an ethidium homodimer. This is an intercalatingdye, which means that it binds to the entire length of the DNA moleculeby inserting itself into the base-stacking zone of the DNA chain. Thedye has a nice property that the fluorescence is greatly enhanced(10,000×) when the molecule is conjugated with DNA, so minimalbackground fluorescence from unbound dye free in solution is produced.For that matter, there is very little dye left in solution, because thebinding is extremely tight between the dye and the DNA molecule. YOYO-1excites near 490 nm wavelength, and emits near 514 nm. The insertion ofthese dyes tends to increase the contour length of the molecule. Adye-to-base-pair ratio of 1:10 is used in one embodiment.

The DNA is stained by first preparing a mixture of YOYO-1 dye and the5×TBE buffer solution and about 3\% vol/vol. of β-mercaptoethanol. Thelatter is a reducing agent which scavenges oxygen radicals from thesolution, thereby reducing the rate of photobleaching of the dye. Thedye, 5×TBE and the β-mercaptoethanol are mixed together and then the DNAis added afterwards. A good value of concentration for working withnanofluidic devices seems to be in the range of 10 uM DNA base-pairs. Toachieve the 1:10 dye-to-base-pair ratio for this concentration of DNA, 1uM YOYO-1 is used. The DNA is allowed to sit for 1 hour at roomtemperature, and then incubated at 40° C. for 10 minutes. The DNAsolution is then ready to be loaded into the devices.

Reservoirs 430 and 435 for fluid and electrodes are attached to thefront face of barrier 415 using silicone RTV elastomer. The resevoirsare cylindrical sections of polypropylene tubing about 2.5 mm in length.This length allows a microscope objective to clear the reservoirs andstill focus on the surface. A small hole about ½ mm is drilled in theside of each reservoir, and a platinum electrode 440,445 is insertedthrough the hole into the interior of each reservoir.

The sample to be analyzed is injected into one of the reservoirs, and aglass cover 430,435 approximately 0.1 mm thick and 4 mm×4 mm square issmeared with vacuum grease and fastened to the top of the reservoir toprevent evaporation. The liquid fills the devices by capillary action,so no special measures are required except to wait approximately 10minutes before filling the reservoir at the opposite to prevent trappingof bubbles in the center of the device.

Occasionally bubbles may be trapped in inconvenient places, and in thesecases, the bubbles are removed by a combination of electric fields andalternating warm and cool ambient temperatures. Degassing is a procedurefor removing dissolved gas by putting the liquids in a chamber at undervacuum. Use of degassing small volumes of liquid can speed re-absorptionof trapped gas.

Typical runs use a pulse duration varying from 0.1 seconds up to 8seconds. The recoil time allowed is increased as a multiple of the driveduration. Though according to theory, the recoil time is expected to goas the square of the drive duration, the program of recoil durationsused is always above the required duration to minimize impact on thedata.

In FIG. 5, an alternative free energy barrier is a membrane 510 shown inperspective with a portion in cross section. The membrane has aplurality of pores 520, one of which is shown in cross section at 530with a molecule 540 shown partially into pore 530. In some embodiments,the pore size varies between 15 nm and 1 um. For smaller pores, thethickness of the membrane is approximately 6 um, and may be thicker forlarger pore size, such as a thickness of between approximately 20-30 um.Smaller size pores may also work, but perhaps not quite as well Thedensity of the pores may be varied as desired given structural integritylimitations of the membrane.

Such membranes are commercially available and are manufactured bytechniques such as the track-etch technique known in the art of membranefilter manufacture. In this technique, a polycarbonate membrane issubjected to bombardment by high-energy ionic nuclear fragments. Thesefragments cause chemical changes to the polymer matrix in the vicinityof the path along which the ion travels. These chemical changes changethe solubility of the matrix in some etchants, such as potassiumhydroxide, allowing the tracks to be developed into small holes. Theseholes can be made very regular in size and shape, and can range in sizefrom 10 nm in diameter or smaller up to several microns. In the presentembodiment, the membrane is contacted with the medium in which themolecules are suspended and the force on the molecules is directedperpendicular to the membrane. The free-energy barrier is formed by thelower entropy of molecules situated inside the pores in the membrane. Afurther embodiment is several of these membranes stacked together toform an array of juxtaposed barriers in which separation occurs. Thisembodiment involves some contrivance to prevent the membranes frombecoming stuck together. This can be an open fabric of fine threadsseparating adjacent membranes, or it can comprise surface texuremodification of the membrane itself to restrict contact between adjacentmembranes to those areas elevated by the texturing.

A further embodiment of a membrane 610 serving as a free energy barrieris shown in FIG. 6. In this embodiment, the membrane has similarparameters as membrane 510, but in addition to pores 620, also has aplurality of surface features, such as bumps 630 to prevent contactbetween adjacent structures, such as further membranes. In oneembodiment, the surface features are formed when the membrane is cast. Asurface on which a film for the membrane is cast, contains dimplescorresponding to the surface features. In further embodiments, thefeatures may be ridges or other structures providing the ability toseparate the membrane from other structures. An alternative techniquefor making the membrane film comprise embossing using well knowntechniques commonly used in the manufacture of compact discs.

Bumps 630 are spaced approximately 2 um apart in one embodiment, with aheight approximately 1 um tall. In further embodiments, the bumps 630are approximately 5-6 um tall, and approximately 20-30 um apart. Instill further embodiments, both sides of the membrane contain bumps.

FIG. 7 illustrates a plurality of juxtaposed adjacent membranes 710,720, 730, 740 and 750. Each membrane has a plurality of pores 760 andbumps 760. The membranes are spaced at least approximately twice thediameter of the pores to ensure acceptable entropic confinement. If tooclose, there is no entropic barrier. If further apart, more time isrequired for molecule drift to move molecules through the membranes.

In one embodiment of the invention, successive membranes are rotatedwhen assembled to ensure that bumps do not mate, and reduce the distancebetween adjacent membranes. In further embodiments, at least twodifferent patterns of bumps are available for alternating membranes toensure proper distance. In yet further embodiments, no bumps arerequired, and the membranes are held in place by suitable framespositioned between reservoirs. Such membranes are thick enough, andseparated far enough to ensure flexing of membranes does not bring themtoo close together.

In the normal operation of the method of this invention, the longestlength of molecule that can be effectively separated is related to thelongitudinal dimension of the high free-energy region of the device. Forexample, if the membrane embodiment is considered, and the membrane hasa thickness of 20 microns, the longest strand of DNA that could beeffectively separated will be some multiple of 20 microns, depending onthe degree of elongation of the molecule as it passes through thehigh-free energy region. Typical values of this multiple will be between1 (in the limit of extreme elongation) to 5 (for weakly elongatedmolecules). This provides a performance limitation in cases where thelongitudinal dimension of the high-free energy region is limited.

FIG. 8 provides a time sequence illustration of an alternative electricfield pulsing algorithm using a membrane 10 microns in thickness, and aDNA molecule 50 microns in length. An electric field is applied at 810,causing both a short molecule 812 and a long molecule 814 to enterrespective pores. In this system, the leading end of the molecule wouldemerge from the downstream side of the high free-energy region beforethe trailing end of the molecule would clear the low free-energy regionon the upstream side, as is required to discriminate lengths.

When the field is removed, the molecule enters a metastable state—i.e.the total free energy is elevated by the occupancy of the molecule inthe high free-energy region but there is no impetus to recoil because asmall change in position of the molecule does not change the energy ofthe molecule. This state is illustrated at 820. In this configuration,movement causes DNA to be drawn from one of the low-energy regions inorder to deliver it to the other for which there is no net change inenergy.

One method for overcoming this limitation is to apply a drive pulse asdescribed herein and allow the molecules to pass through the meta-stablestate as described above. In this case, for a given pulse duration, along molecule might remain in the metastable state at the end of thepulse, while a shorter molecule will have passed through the metastablestate, and accumulated entirely on the downstream side of the highfree-energy region as indicated at 820. Now, the longer molecule willnot recoil on its own, so a pulse of retrograde force is applied belowthe critical force required to initiate passage into the highfree-energy region as illustrated at 830. The shorter moleculecompletely on the downstream side of the high free-energy region remainsin that region for lack of sufficient force to insert it into the highfree-energy region. The longer molecule straddling the high free-energyregion will move readily because no energy change is required for itsmotion. This state is illustrated at 840. The retrograde pulse must besufficiently long to completely recoil all molecules in the metastablestate to the upstream side of the high free-energy region. In thismanner the same conceptual mechanism can be applied in the absence of along high free-energy region. This method has an additional advantagethat the adverse scaling of recoil time with molecule length iseliminated. In this case the recoil time scales linearly with themolecule length. As before the process can be repeated with the same ordifferent pulse programs to continue to separate in the same region oflengths or different regions of length. Also as before, many barrierscan be presented sequentially to enhance the resolution of thetechnique.

CONCLUSION

Separation of long molecules by length is obtained by forcing suchmolecules to traverse a boundary between a low free-energy region and ahigh free-energy region. In one embodiment, the high free-energy regionis a dense pillar region or other structure formed on a semiconductorsubstrate. The low free-energy region is a larger chamber or structureformed adjacent the high free-energy region. Other structures and forcebarriers may also be used as is apparent from the description. Further,while an electric field is pulsed to provide the force on the molecules,other forces may also be used consistent with the force barrier, such asgravity, pressure and centrifugal force.

Structures for practicing the invention were also described. It shouldbe noted that the specifications thereof were highly dependent on thesize of the molecules, and that they will change for different sizedmolecules. Further, the specification may also be changed for operationon the same sized molecules as described above to while still obtainingbenefits provided by the invention.

1. A method of separating molecules by length, the method comprising:applying force to move molecules from a region of low free-energy into aregion of high free-energy; and removing the force to cause moleculesnot entirely within the region of high free-energy to extract themselvesback into the region of low free-energy.
 2. The method of claim 1wherein the force comprises an electric field pulse sufficient to causethe molecules to move into the region of high free-energy.
 3. The methodof claim 1 and further comprising prior to applying force, applying agathering force that is less than the force required to cause themolecules to move into the region of high free-energy.
 4. The method ofclaim 1 wherein the difference in free-energy between the regions is dueto differences in entropy for molecules present within them.
 5. A methodof separating molecules by length, the method comprising: forcingmolecules to a boundary between a region of high entropy and a region oflow entropy; applying high field pulse to drive molecules into the lowentropy region; and allowing molecules not driven entirely within thelow entropy region to recoil into the high entropy region.
 6. The methodof claim 5 wherein a subcritical field is used to force molecules to theboundary.
 7. The method of claim 5 wherein the pulse is a super criticalvalue to start driving molecules into the low entropy region.
 8. Themethod of claim 5 wherein the low entropy region comprises a densepillar region.
 9. The method of claim 5 wherein the elements areiteratively repeated.
 10. The method of claim 5 wherein the elements areiteratively repeated with progressively longer duration or highervoltage pulses.
 11. The method of claim 5 wherein the elements areiteratively repeated with varying duration and voltage pulses.
 12. Themethod of claim 5 wherein recoil is aided by applying an alternatingcurrent field.
 13. A separator for separating molecules of varyinglength, the separator comprising: a high entropy region; a low entropyregion having an entropic barrier between the high entropy region andthe low entropy region; and a field generator that generates a pulse ofentropic force greater than the entropic barrier to drive shortermolecules entirely into the low entropic region while longer moleculesare only partially driven into the low entropic region.
 14. Theseparator of claim 13 wherein the field generator generates electricfield pulses of selectively varying voltage and duration.
 15. Theseparator of claim 13 wherein the field generator also generates asubcritical field to push molecules toward the entropic barrier prior togenerating the pulse of entropic force greater than the entropicbarrier.
 16. The separator of claim 13 wherein the low entropy regioncomprises a dense pillar region.
 17. The separator of claim 13 whereinthe low entropy region comprises a membrane penetrated by nanoscaleholes and the high entropy region comprises space outside the holes. 18.The separator of claim 17 wherein the membrane further comprises aplurality of features extending from the membrane.
 19. The separator ofclaim 18 wherein the features comprise bumps.
 20. The separator of claim13 wherein a plurality of regions of high and low free-energy arejuxtaposed within the separator in a manner that faciltates moleculesthat have been separated by one boundary to be further separated by oneor more additional boundaries.
 21. A method of separating molecules bylength, the method comprising: applying force to move molecules from aregion of low free-energy into a region of high free-energy; andapplying a retrograde force to cause molecules still partially withinthe low free-energy region to extract themselves back into the region oflow free-energy.
 22. The method of claim 21 wherein the retrograde forceis below a critical force required to initiate passage of the moleculesinto the high free-energy region.
 23. The method of claim 21 wherein theforces comprise electric fields.
 24. The method of claim 21 whereinmolecules having portions within either side of the high free-energyregion are straddling the high free-energy region, and wherein theretrograde force is of sufficient duration to cause straddling moleculesto completely extract themselves back into the region of lowfree-energy.
 25. A separator for separating molecules of varying length,the separator comprising: a plurality of high free-energy regions; aplurality of low free-energy regions juxtaposed between the highfree-energy regions; and a force generator that generates a pulse offorce to drive molecules into the high free-energy regions.
 26. Theseparator of claim 25 wherein the force generator generates a retrogradepulse not sufficient to drive molecules into the high free-energyregions.
 27. The separator of claim 25 wherein the high free-energyregions comprise nanoscale pore membranes.